The overall goal of this proposal is to develop chemically defined reagents that support hematopoietic stem cell proliferation and differentiation. Such reagents would be valuable in hematological research, especially for bone marrow transplantation, gene therapy and purging. During the Phase I effort, we have developed reagents that are composed of chemically defined components, free of any proteins derived from human sources which strongly support CD34+ cell proliferation and differentiation. We propose in the Phase II effort to optimize these reagents and culture conditions for ex vivo culture of bone marrow, cord blood and mobilized peripheral blood CD34+ cells. The Phase II effort will focus on cytokine mixtures, concentration effects and culture time frames with respect to total cell number, CFU-GM formation, cell cycle kinetics, and immunophenotype analysis. The short-term and long-term reconstituting ability of the cultured cells will be assessed using the large animal in utero sheep model for human hematopoiesis. Such studies will be invaluable in developing reagents that are chemically defined, and most importantly for removal of proteins derived from human blood components that are subject to various diseases and thus FDA recall. These reagents will foster the development of clinical protocols for the treatment of disease using cellular therapies. PROPOSED COMMERCIAL APPLICATION: Defined reagents that support stem cell expansion will be valuable to the researcher and clinician for gene therapy, bone marrow transplantation and purging.